Brooks C, Ergul A
Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
J Mol Endocrinol. 1998 Dec;21(3):307-15. doi: 10.1677/jme.0.0210307.
Big endothelin-1 (big ET-1) is converted to 21-amino acid residue endothelin-1 (ET-1) via a specific cleavage at Trp21-Val22 by endothelin converting enzyme (ECE). This conversion is an essential step to produce bioactive ET-1 and represents a regulatory site in the biosynthesis of this potent vasoconstrictor. ECE-1a, a unique membrane-bound enzyme, processes big ET-1 more efficiently than other big ET isoforms, which mainly differ in the C-terminal tail (residues 22-38). In this study, each of the highly conserved residues, Val22, Pro25, Pro30, Gly32, Leu33, and Gly34 were replaced with Ala in the preproendothelin-1 (PPET-1) cDNA using site-directed mutagenesis. The mutant and wild-type cDNAs were transiently transfected into Chinese hamster ovary cells along with ECE-1a cDNAs, and concentrations of the resulting recombinant peptides, ET-1 and big ET-1, in the transfection media were then measured. The concentration of immunoreactive ET-1 in the media from Val22, Pro25, Pro30, Gly32, and Leu33 mutant PPET-1-transfected cells was 4- to 6-fold lower than that of wild type and (Gly34-->Ala)PPET-1. Moreover, with the exception of Gly34 there was a corresponding increase in the concentrations of immunoreactive big ET-1 in the media from mutants. Similar results were obtained when His27, Val28, and Ser35 of big ET-1( )were substituted with the corresponding residues in big ET-2 and big ET-3. These findings suggest that the C-terminal tail has an important role in the intracellular processing of big ET-1 by ECE-1a. Herein we also report that a recombinant big ET-1 analog we previously generated and characterized, (Ala21)big ET-1, inhibits ECE-1a activity in a dose-dependent (K i=1 microM) and competitive manner.
大内皮素 -1（big ET -1）通过内皮素转化酶（ECE）在Trp21 - Val22处的特异性切割被转化为21个氨基酸残基的内皮素 -1（ET -1）。这种转化是产生生物活性ET -1的关键步骤，并且代表了这种强效血管收缩剂生物合成中的一个调节位点。ECE -1a是一种独特的膜结合酶，比其他主要在C末端尾巴（残基22 - 38）上存在差异的big ET同工型更有效地加工big ET -1。在本研究中，使用定点诱变将前内皮素 -1（PPET -1）cDNA中高度保守的残基Val22、Pro25、Pro30、Gly32、Leu33和Gly34中的每一个都替换为Ala。将突变型和野生型cDNA与ECE -1a cDNA一起瞬时转染到中国仓鼠卵巢细胞中，然后测量转染培养基中产生的重组肽ET -1和big ET -1的浓度。来自Val22、Pro25、Pro30、Gly32和Leu33突变型PPET -1转染细胞的培养基中免疫反应性ET -1的浓度比野生型和（Gly34→Ala）PPET -1低4至6倍。此外，除了Gly34之外，突变体培养基中免疫反应性big ET -1的浓度相应增加。当big ET -1（ ）的His27、Val28和Ser35被big ET -2和big ET -3中的相应残基取代时，也获得了类似的结果。这些发现表明C末端尾巴在ECE -1a对big ET -1进行细胞内加工过程中具有重要作用。在此我们还报告，我们之前生成并表征的一种重组big ET -1类似物（Ala21）big ET -1以剂量依赖性（K i = 1 microM）和竞争性方式抑制ECE -1a活性。
